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Multiplex Quantification of 12 European Union Authorized Genetically Modified Maize Lines with Droplet Digital Polymerase Chain Reaction David Dobnik,*,†,∥ Bjørn Spilsberg,‡,∥ Alexandra Bogožalec Košir,†,§ Arne Holst-Jensen,‡ and Jana Ž el† †
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia Section of Virology, Norwegian Veterinary Institute, P.O. Box 750, Sentrum, 0106 Oslo, Norway § Josef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia ‡
S Supporting Information *
ABSTRACT: Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/ endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.
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considered individually or food consisting of a single ingredient...” Thus, the labeling threshold is on a per ingredient basis, not on a per GMO basis. In analytical terms, ingredient in this context is interpreted as species.3 So, it is the cumulative concentration, e.g., of all authorized maize GMOs relative to the total quantity of maize that determines if the threshold is exceeded or not. The technical guidance for sampling and detection of GMOs4 states that the GMO content should be expressed as ratio of GM part in relation to the taxon specific part, meaning that GMO content can either be measured as additive concentration of individual authorized events or it can be measured as the total concentration of this group directly. The latter appears as a more cost-efficient approach, but hitherto it has not been possible to implement due to lack of suitable analytical methods.
any countries in the world regulate the cultivation and trade with genetically modified organisms (GMO).1 Most commonly there is an approval system and mandatory labeling above a certain threshold. The enforcement of such regulations depends on the ability to detect and quantify the presence of GMOs in food, feed, and seed products. In the European Union (EU) this is enforced for all authorized GMOs through Regulation (EC) 1829/2003.2 This regulation specifies that the developer of a GMO has to provide a method for quantifying the specific GMO event and funding for a collaborative trial to validate the method. Quantification of the GMO is done relative to a species specific reference gene. Thus, there exists a set of validated event specific GMO detection methods (available at http://gmo-crl.jrc.ec.europa. eu/gmomethods/). These are all standard curve based real-time PCR (qPCR) methods. In Europe, the labeling threshold for authorized GMO events is specified in Regulation 1829/2003.2 This regulation states that labeling: “shall not apply to foods containing material which contains, consists of or is produced from GMOs in a proportion no higher than 0.9% of the food ingredients © 2015 American Chemical Society
Received: March 17, 2015 Accepted: July 13, 2015 Published: July 13, 2015 8218
DOI: 10.1021/acs.analchem.5b01208 Anal. Chem. 2015, 87, 8218−8226
Article
Analytical Chemistry
explain the conversion of copy number ratio to mass fraction. For the purpose of this manuscript, the conversion factor set in EURL Technical Guidance 619/201119 was used. Presently, two designated dPCR technologies are available: microfluidic/chip based dPCR and emulsion (droplet) based dPCR.20 Several studies have tested the applicability of dPCR for GMO detection.21−25 The practicability and applicability of using the droplet dPCR (ddPCR) in routine GMO diagnostics was recently shown for duplex reactions.24 In the present study it was explored whether ddPCR could be multiplexed to a high degree and utilized to quantify a group of GMOs for compliance with the EU labeling regulation.2 The analytical procedure of GMO testing include sample preparation, DNA extraction and purification, (q)PCR amplification, and data evaluation. These steps are nowadays often referred to as modules.5 There are several advantages of using the modular approach in GMO testing, especially it is more favorable when the methods are being validated.26 This study was focused only on the module of PCR amplification and does not include the evaluation of other modules. To be able to analyze GMO content per ingredient in a cost efficient manner and in line with the EU regulatory requirements,2 a set of validated qPCR modules was used and combined for multiplex ddPCR. In this manuscript, the data on the performance of the developed assays are presented and their practicability, applicability, and potential impact is discussed.
Standard curve based qPCR is sensitive and robust and is regarded as the gold standard for GMO analysis. As the number of authorized GMOs has increased over time, the required number of qPCR analyses has increased correspondingly. An analysis regime based only on separate event specific qPCRs is no longer cost efficient. As a more cost efficient approach, many laboratories now employ one of several alternative screening approaches, testing for presence of genetic elements commonly found in GMOs.5 The observed presence/absence pattern for each sample can be compared with a reference table of presence/absence of the corresponding elements in known GMOs, e.g., using the GMOseek software.6,7 At and above the limit of detection (LOD), the presence of GMOs containing elements that are not observed in the sample can effectively be excluded. The list of remaining candidate GMOs after screening serves as a starting point for quantification and confirmation of events present in the sample. Recently a multiplex version of one of these screening approaches8 was developed as a single qualitative pentaplex PCR.9 Multiplex PCR analysis obviously has the potential to improve the cost efficiency, but a multiplex PCR is a complex experimental system with potential interference between oligonucleotides and amplification products (target and nontarget). The development of quantitative methods to a high level of multiplexing has proven difficult. Although, several multiplex qPCRs exist, only two (duplex) are interlaboratory validated: one in Europe10 and one in Japan.11 Digital PCR (dPCR) as a concept was invented in 1992.12 In dPCR the reaction mixture is distributed into a large number of partitions, where each partition contains zero, one, or more copies of the target nucleic acid. Positive and negative signal in partitions is scored at end point of the PCR and the initial DNA concentration can be calculated using the Poisson distribution.13 Digital PCR does not depend on PCR efficiency for DNA quantification to the same degree as standard curve based qPCR. Partial inhibition of a PCR will not affect quantification with dPCR as only discrete data on signal and not amplitude of the signal will be used in the calculations. Digital PCR can be performed in normal 96-well PCR plates using fluorescence probes,14 and different approaches have been explored for the use in GMO analysis.15,16 Although one of these was formally collaborative trial validated as a simplex dPCR approach for quantitation of GTS 40-3-2 (RoundupReady) soya (Marco Mazzara et al., personal communication), this experimental setup did not improve cost efficiency significantly due to large chemical consumption and low statistical power. The dPCR is measuring the absolute number of copies in the sample; therefore, the final result for GM content is given as copy number ratio of GM event and taxon specific gene. Most widely used reporting unit for qPCR is nevertheless the mass ratio, as the majority of reference materials are certified for the GM% in mass. Thus, although the PCR reaction is amplifying the actual DNA copies, the final result is converted in mass ratio based on the standard curve of reference material. This fact made the topic unclear already from the beginning. The official unit of measurement has however never been exactly defined for the whole GMO field but rather for individual scopes (prepackaged food,17 EU unauthorized GMOs18 and recently for reporting of proficiency test results [EURL-GMFF Invitation letter, 2015]). As the EU Commission is apparently headed toward accepting only mass fraction as the accepted unit of measurement, an ENGL working group on the Unit of measurement has been recently established, who will try to
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EXPERIMENTAL SECTION Test Material. A list of all CRM test materials is provided in Table S-15 the Supporting Information. They all have certified mass/mass (m/m) GM maize/wild-type maize material ratios. Routine diagnostic samples containing maize events (seeds or flour) and samples from proficiency programs (USDA and EURL) were also used in this study (Table S-12 in the Supporting Information). A specificity study was conducted on samples containing either GM maize events not authorized in the EU, DNA from non-GM maize/sweet corn, and GM and non-GM soybean, rape seed, rice, and linseed samples without maize (Table 2). DNA Extraction. DNA was extracted and purified from 200 mg of starting material for all samples using a cetyltrimethylammonium bromide (CTAB) protocol with RNase-A solution and proteinase-K solution for removal of RNA and proteins from the sample (as described in annex A.3 of ISO 21570:200527), with small adaptations. Dilutions of the extracted stock DNA solutions were made in nuclease- and protease-free water (Sigma-Aldrich Chemie GmbH, Munich, Germany). All samples were stored at −20 °C. Primers and Probes Mix Preparation. The hmgA gene was used as the endogenous reference gene for maize, to which relative quantity of the GM events was estimated. For quantification of GM maize lines, the construct (for Bt11) or event (all others) specific qPCR modules were used. Nucleotide sequences of probes and primers used are presented in Tables S-4 and S-5 in the Supporting Information. The GM content in each sample was determined by simplex ddPCR. A total of 13 primer and probe sets were prepared for simplex reactions targeting individual events, constructs or the endogene, as well as 12 sets for duplex reactions where primers and probes for endogene (hmgA) and individual events or construct were mixed. The preparation of 10-plex and 4-plex primer and probe (PPP) mixes was done as follows. For 10plex, primers and probes for the endogene (hmgA) and nine 8219
DOI: 10.1021/acs.analchem.5b01208 Anal. Chem. 2015, 87, 8218−8226
Article
Analytical Chemistry
copies of transgenes covered by 10-plex and from 1734 to 0.002 copies of transgenes covered by 4-plex. The dilution series for both multiplexes contained from 139 302 to 0.2 copies of the endogene hmgA. Twelve replicates of the dilution series were measured by ddPCR (three separate runs in 3 days each containing four replicates). Another dilution series was prepared in terms of transgene content, where the dilutions of DNA mix 1 for 10-plex contained 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 0.9, and 2% of all transgenes found in 10-plex, and 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, and 0.9% of all transgenes found in 4-plex for DNA mix 2. Six replicates of these dilution series were measured by ddPCR. The absolute/relative (copy number/%) limit of quantification (a/rLOQ) and absolute/ relative limit of detection (a/rLOD) for ddPCR were determined based on these experimental results. The LOQ was determined as the aLOQ or rLOQ in the sample, where the relative standard deviation (RSD) of all replicates was below 25%. The LOD was determined as the aLOD or rLOD in the sample, where all replicates still produced a positive signal. Specificity. To test specificity the DNA extracts from 12 different samples (3 GM maize events not authorized in the EU, 2 GM soybean events, one GM rice event, one GM oilseed rape event and five different nontransgenic samples; Table 2) were tested with the 4- and 10-plex ddPCR assay. Reactions were performed in duplicates per sample per assay. Fitness for Purpose. To assess whether the new assays are fit for purpose, 13 different samples from proficiency programs (USDA and EURL) and four routine diagnostic samples (Table 3) were tested with both multiplex assays. To obtain a robust mean value of GM content, the proficiency test reports were used to make an average of all reported values per event, then to calculate the sum of all present events, and finally to calculate standard deviation and Z-score values. For the results to be directly comparable, the conversion of ddPCR results (measured as GM% in copy number ratio) into the mass ratio (GM% in mass fraction) was done according to EURL Technical Guidance 619/2011.19 For sample 189/09, containing GM maize in which the transgene originated from the male parent, another conversion using the factor 0.36 (based on information from Holst-Jensen et al.3 and our previous ddPCR analyses of those GM lines [data not shown]) was also performed. For routine diagnostic samples, the additive concentration of GM maize events was calculated based on results of quantification with qPCR performed in the NIB laboratory for official control under ISO17025 accreditation. Robustness. The robustness of the ddPCR multiplex assay was evaluated by making small modifications to the protocol. These included (1) use of a PCR cycler with slower ramp rate (GeneAmp 9700 PCR cycler [Applied BioSystems, Foster City, CA]), (2) different annealing temperatures (61 and 59 °C), (3) use of another Mastermix (2xddPCR Supermix for probe containing dUTP [Bio-Rad, Pleasanton, CA]), (4) pipetting all eight reaction mixes to the cartridge in one step with a multichannel pipet, and (5) transfer of generated droplets to PCR plate with an automatic electric multichannel pipette. Two samples from the repeatability experiments (DNA mix, 60× and 120× dilution) were used in the robustness experiments, and the results for normal conditions were taken from the repeatability experiments. The 60× sample was tested in duplicates with both multiplex assays, whereas only one reaction was tested for the 120× sample per multiplex assay.
events, GA21, MON810, MON863, DAS1507, MIR604, MON88017, MON89034, MIR162, and T25, were mixed (for final concentrations see Table S-4 in the Supporting Information). For 4-plex, primers, and probes for the endogene (hmgA), two events, DAS59122 and NK603, and one construct, Bt11, were mixed (for final concentrations see Table S-5 in the Supporting Information). Primers and probes were purchased from Eurofins MWG Operon (Ebersberg, Germany) or from Integrated DNA Technologies (Leuven, Belgium). Primers and probes were shipped lyophilized and diluted in nuclease- and protease-free water (Sigma-Aldrich Chemie GmbH, Munich, Germany) upon reception. Droplet Digital PCR Reactions and Data Analysis. The reaction mix for all reactions, simplex as well as multiplex, was composed of 10 μL of 2xddPCR Supermix for probe (No dUTP) (Bio-Rad, Pleasanton, CA), 6 μL of primer and probe mix, 4 μL of DNA template (or 4 μL of nuclease- and proteasefree water for nontemplate control), mixed into a 20 μL reaction volume. Droplets were generated in 8-well cartridges, using the QX100 droplet generator (Bio-Rad, Pleasanton, CA). Water-in-oil emulsions were transferred to a 96-well plate and amplified in a T100 PCR cycler (Bio-Rad, Pleasanton, CA). Thermal cycling conditions were 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles of a two-step thermal profile comprising of 15 s at 95 °C and 60 s at 60 °C at ramp rate 2.5 °C/s. After cycling, each sample was incubated at 98 °C for 10 min and then cooled to 4 °C. Plates were then transferred to the QX100 droplet reader (Bio-Rad, Pleasanton, CA). Data acquisition and analysis was performed using QuantaSoft (BioRad, Pleasanton, CA) software. Positive droplets, containing amplification products, were discriminated from negative droplets without amplification products by applying a fluorescence amplitude threshold (Figure S-1 in the Supporting Information). The threshold was set manually, using both the fluorescence amplitude vs event number (1D amplitude) and the histogram of events vs amplitude data streams, on each of the FAM and VIC channels (2D amplitude). Data generated by the QX100 droplet reader were rejected from subsequent analysis if a clog was detected by the Quantasoft software or if a low number of droplets (30% for both samples, indicating that the difference may be the result of increased variability between replicates. A significant effect of PCR cycler and multichannel pipetting was observed in only one of two samples (Table 3). A detailed look at the results, comparing the
replicate or (b) as per partition (droplet). Acceptance values of false negative rates ≤5% are widely adopted33 and a false positive rate below 5% of the replicates (wells) is a translation of this threshold. These are also the thresholds proposed in a recently published guidance document.34 In present experiments, a false-positive rate of 0% was observed over all replicates (Table S-11 in the Supporting Information), showing a good specificity of the assays. When the false positive rate was assessed over droplets in each replicate, the observed false positive rate was below 0.015% (data not shown). The latter satisfies the acceptance criterion for a false positive rate per partition set to
